Hybrid cooperative complexes of hyaluronic acid

ABSTRACT

Describes cooperative hybrid complexes of hyaluronic acid, a simple and economical method for production thereof and use thereof in the area of medicine, cosmetics and food.

FIELD OF THE INVENTION

The present invention relates to the field of products based onhyaluronic acid.

PRIOR ART

Hyaluronic acid, generally indicated hereinafter, together with itssalts, called hyaluronans, as HA, is a negatively charged straight-chainpolysaccharide, made up of a repetition of n disaccharide units(-4GlcUAβ1-3GlcNAcβ1-), in which D-glucuronic acid (GlcUA) andN-acetyl-D-glucosamine (GlcNAc) are joined with alternating β-1,3 andβ-1,4 glycosidic bonds.

HA is a highly water-soluble polysaccharide and solutions of HA displaya non-Newtonian type of viscoelastic behaviour. These properties dependon the molecular weight (and therefore, as HA is a linear polymer, onthe length of the chain), the concentration, the pH and the ionicstrength.

Owing to its biological properties and functions, HA has high addedvalue (its commercial value greatly exceeds that of the other naturalpolysaccharides), with applications that range from the medical sectorto cosmeceuticals and nutraceuticals. Its viscoelastic properties,coupled with the complete absence of toxicity or immunogenicity (thestructure of HA is always the same in all living organisms in which itis present), have led to varied and extensive applications.

In many of these applications the performance depends on the molecularweight of the HA. For this, the average molecular weight of HA and thepolydispersity index Mw/Mn (which measures the width of the curve ofmolecular weight distribution, where Mn is the number-average molecularweight, defined as the total weight of all the polymer molecules in asample divided by the total number of molecules, and Mw is theweight-average molecular weight, which takes into account the varyingmass of the molecules present) must be the gold standards to beconsidered when developing production processes for HA and strategiesfor application.

In particular, the wide variety of biological responses of HA connectedwith its molecular weight now mean that low molecular weight HA (L-HA)and high molecular weight HA (H-HA) must be used in context.

DESCRIPTION OF THE INVENTION

The present invention describes cooperative hybrid complexes betweenL-HA and H-HA, designated with the acronym L/H-HA, theircharacteristics, the production process thereof and use thereof in thearea of medicine, cosmetics and foodstuffs. Weak forces, such ashydrogen bonds or hydrophobic interactions, can give rise to very stableinteractions between molecules, if these are of the cooperative type.Cooperativeness develops when it is possible for multiple bonds to formbetween the molecules, and being weak, they break randomly thereafter,but can immediately reform owing to the existence of intact vicinalbonds, which maintain the structural components of the bond at adistance useful for its reformation.

The molecules of HA in solution are characterized by cooperativephenomena of interaction based on formation of hydrophobic bonds andinterchain hydrogen bonds, and the cooperativeness of these interactionsdepends on the length and therefore on the molecular weight of thechains. The long chains of H-HA give stable interactions between them,which involve all the molecules present in solution, giving rise to athree-dimensional network, whereas molecules of L-HA give interactionsthat are less stable, leading to systems of aggregation that do notsimultaneously involve all the molecules present, which instead interactin clusters. This differing mode of aggregation of H-HA and L-HA insolution is responsible for the large differences in rheologicalbehaviour, such as for example the viscosity of solutions of HA, whichis a very important property for numerous applications, especially inthe medical field.

The rapid drop in viscosity of solutions of HA as a function of themolecular weight in fact actually depends on this varying capacity forintermolecular interaction, so that, at equal concentration, solutionsof H-HA with molecular weight above 1·10⁶ Da have viscosities of higherorders of magnitude than those of solutions of L-HA with molecularweight between 5·10³ and 5·10⁵ Da. Owing to the strong cooperativenessof the interactions between the long chains of H-HA, when L-HA isdissolved in a viscous solution of H-HA, in the short term nosignificant differences in viscosity of the resultant solution areobserved, indicating that the two molecular populations behaveindependently and that the formation of L/H-HA cooperative hybridaggregates is a thermodynamically unfavourable process. Only with thepassage of time (days—weeks), a slow but constant decrease in viscosityis observed that cannot be attributed to hydrolytic processes, and thiseffect is more evident if the molecular weight of the L-HA is less than10⁵ Da. The continual variation of the physicochemical properties, inparticular the viscosity, of these solutions makes them unsuitable forpractical applications, which instead require constant rheologicalcharacteristics.

It is therefore clear, in view of the foregoing, that cooperative hybridcomplexes between hyaluronic acid of low molecular weight and hyaluronicacid of high molecular weight, having properties that allow them to beused for the desired purposes, are not currently available.

Now, it was found, surprisingly, that it is possible to create stableL/H-HA cooperative hybrids by submitting aqueous solutions containingH-HA and L-HA together to a suitably configured thermal cycle.

The solutions of stable L/H-HA cooperative hybrids according to theinvention are characterized by viscosities that do not change over timeand that are notably lower than before the thermal cycle.

Such behaviour cannot be ascribed merely to a process of thermaldepolymerization of HA.

There are four parameters that critically determine the formation of theL/H-HA complexes and their rheological properties:

-   -   a) the simultaneous presence of the two types of HA (L-HA and        H-HA) in the same solution;    -   b) the molecular weight of the two species of HA used in the        process of formation of the L/H-HA hybrid system;    -   c) the relative proportions of the two species of HA used;    -   d) the profile of the thermal cycle to which the solution is        exposed.

The simultaneous presence of the two types of HA (L-HA and H-HA) in thesame solution is a necessary condition because when the solution,submitted to the thermal cycle, reaches a high enough temperature,energy conditions are created that are able simultaneously to ruptureall the interactions between the chains of H-HA and those between thechains of L-HA, and in these conditions the prerequisites no longerexist because the weak interactions that develop between the moleculesin solution are of the cooperative type and the polymer chains behave asindependent entities. Next, when the solution is cooled within the scopeof the thermal treatment cycle, interchain interactions begin to reformincreasingly, which in this case develop randomly between all themolecules of HA present in solution, both of high and of low molecularweight, giving rise to hybrid systems, which are stabilized when, withincreasing number of weak intermolecular bonds, their cooperativenessmeans that the mode of interaction that has developed between thepolymer chains of different molecular weight does not change over time.Confirmation of the validity of this mechanism is the fact that, onsubmitting two solutions, one of L-HA and one of H-HA, separately to thethermal cycle and then mixing them together after cooling, at equalconcentration of the species in solution, we do not observe the dramaticand immediate decrease in viscosity attributed to formation of thehybrid system, which can only form if the two molecular species arepresent simultaneously during the thermal cycle.

The molecular weight of HA used in constructing L/H-HA hybrid systemscritically determines their rheological characteristics; the greater thedifference in molecular weight between the L-HA and H-HA used, thegreater, at equal concentration, is the decrease in viscosity of thehybrid system relative to that of the H-HA. Cooperative hybrid L/H-HAcomplexes, characterized by a decrease in viscosity, can be obtained ifthe molecular weight of the L-HA is between 1·10⁴ and 1·10⁶ Da and thatof the H-HA is given by the formula MW_(H-HA)≧MW_(L-HA)/0.9.

The relative proportions of L-HA and H-HA, determining the stoichiometryof the hybrid, contribute to modification of their rheologicalproperties relative to the species hybridized between them; the decreasein viscosity due to formation of the complex increases with increase ofthe L-HA/H-HA stoichiometric ratio used. Normally said ratio is between0.1 and 10, preferably from 0.5 to 2.

The complexes according to the invention normally have a viscosity from1.1 to 200-fold less than that of a solution containing the H-HAhyaluronic acid alone used for forming the complex

The thermal profile that leads to the formation of cooperative hybridL/H-HA systems starting from solutions containing L-HA and H-HAenvisages that the solution is first heated to temperatures between 80and 160° C., preferably between 100 and 120° C. and then cooled rapidlyto room temperature. The L/H-HA hybrid systems thus obtained are stableover time, attesting to maintenance of their rheologicalcharacteristics.

As already mentioned, the solutions of L/H-HA hybrid complexes accordingto the present invention can easily be obtained by mixing aqueoussolutions of H-HA and L-HA of desired molecular weight and submittingthe resultant solution to the thermal cycle indicated above; preferablythe concentration of the solution of L-HA is between 0.01 and 50% w/wwhile that of the solution of H-HA is between 0.01 and 10% w/w.

Cooperative hybrid L/H-HA complexes in the solid state can be obtainedfrom solutions containing them in various ways:

-   -   a) by precipitation of the solutions containing them, by adding        water-miscible organic solvents, such as low molecular weight        alcohols, acetone, etc.;    -   b) by solvent evaporation;    -   c) by spray-drying;    -   d) by lyophilization.

Cooperative hybrid complexes similar to those described above,characterized by low values of dynamic viscosity, can moreover beobtained by high-temperature thermal treatment of aqueous solutions ofH-HA with low molecular weight polysaccharides, such as chondroitin,chondroitin sulphate, dextrins, cyclodextrins, dextrans.

The cooperative hybrid L/H-HA complexes are, because of theirrheological characteristics, of considerable interest in some biomedicalapplications, for example: biorevitalization of the skin by intradermalinjections of HA; techniques of viscosupplementation for resolvingpathological situations connected with inflammatory disorders of thejoints; intra-bladder treatment of cystitis; treatment of vaginalinflammatory diseases; treatment of alveolar diseases; treatment of oraldiseases.

The most important advantage connected with the use of cooperativehybrid L/H-HA complexes is their low viscosity, which in medicalpractice allows the use of solutions of higher concentration, but stillsufficiently fluid to be injected with small-bore needles and cathetersor to be nebulized.

Once in contact with the biological environment, the cooperative hybridL/H-HA complexes behave as systems for slow release of L-HA and H-HA,because the chemical complexity of the microenvironment, characterizedby the presence of other species in solution and the enormous surfacesof the cellular structures, permit gradual resolution of theintermolecular interactions that characterize the complex, making bothL-HA and H-HA available in context ab initio, molecular species that invivo have differentiated roles, L-HA that of signalling by interactionwith receptors present on the cell surfaces and H-HA as a fundamentalconstituent of the extracellular matrix.

Non-limiting examples are given below, describing the production,characteristics and use of the cooperative hybrid L/H-HA complexes.

EXAMPLE 1 Production of Cooperative Hybrid L/H-HA Complexes at DifferentTemperatures

Two aqueous solutions of H-HA (MW 1.4·10⁶ Da; Mw/Mn 1.5) and L-HA (MW3.3·10⁴ Da; Mw/Mn 1.8) were prepared at 2% w/v in distilled water, whichare used for preparing the various solutions given in Table 1.

These solutions, containing L-HA and H-HA simultaneously, are submittedto a thermal cycle in autoclave that envisages: a) a heating phase from25° C. up to a maximum temperature in 10 min; b) maintaining thistemperature for a specified period of time (10 min or 40 min); c)cooling the solution to 25° C. in 10 min.

MW and polydispersity index Mw/Mn are determined using a size-exclusionchromatography system equipped with a multidetector, consisting of afour-bridge viscosimeter, a refractometer, a right angle lightscattering detector (RALS) and a low angle light scattering detector(LALS), patented by the American group Viscotek (www.viscotek.com). Thesignal measured with the LALS is proportional to the molecular weightand the concentration, that measured with the viscosimetric detector isproportional to the sample concentration and the intrinsic viscosity,while the refractometer provides measurement of the concentration. TheViscotek apparatus not only makes it possible to determine the molecularweight of HA, but also evaluate the degree of non-uniformity ofmolecular weight in the population of molecules present, described bythe polydispersity index Mw/Mn, automatically calculated by the Viscotekapparatus, and defined as the ratio of the average molecular weight(Mw=Σ_(i)m_(i)M_(i)/Σ_(i)m_(i) where m_(i) is the mass of polymer withmolecular weight M_(i) and Σ_(i)m_(i) is the total mass of polymer, anexpression which, setting m_(i)=n_(i)M_(i) can also be given asMw=Σ_(i)n_(i)M_(i) ²/Σ_(i)n_(i)M_(i)) and weight-average molecularweight (Mn=Σ_(i)n_(i)M_(i)/Σ_(i)n_(i) where n_(i)M_(i) is the mass ofpolymer with molecular weight M_(i) and Σ_(i)n_(i) is the total numberof moles of polymer present). The measurements of dynamic viscosity ηare performed on an Anton Paar Physica MCR 301 rheometer, using ageometry with coaxial cylinders. η is determined at 25° C. at a constantshear rate (γ′=2 s⁻¹) which comes within the range of Newtonianviscosity of the polymer solution (η is constant with respect to γ′ anddepends only on the conformation of the polymer in solution).

TABLE 1 Mixing with thermal cycle- T_(max) (° C.)-time (min) Sol. 2% w/vInitial 120°; 100°; (mL) H₂O solution 10′ 110°; 10′ 10′ 100°; 40′Sample^(o) H-HA L-HA (mL) η (Pa · s) H-HA 100 0 100 21.321 5.632 10.24111.513 5.442 L-HA 0 100 100 0.002 <0.001 <0.001 <0.001 <0.001 L/H-HA 100100 0 19.010 0.038 0.062 0.943 0.051 H-HA + L-HA* 100 100 0 — 4.3349.523 10.530 4.912 *the two solutions at 2% w/v are first treated athigh temperature and then mixed in 1:1 ratio by volume.

Table 1—Measurement of the dynamic viscosity of solutions with aconcentration of 1% w/v of L-HA (MW 3.3·10⁴ Da; Mw/Mn 1.8) and H-HA (MW1.4·10⁶ Da; Mw/Mn 1.5) and of the corresponding stable cooperativeL/H-HA complexes with a concentration of 1% w/v and L-HA/H-HA ratio of1:1 w/w. The thermal treatment cycle in autoclave envisages a heatingphase in 10 min from 25° C. to T_(max), remaining at T_(max) for aspecified time and a cooling phase from T_(max) to 25° C. in 10 min. Themeasurements of η are taken immediately after the thermal treatment. Thedata in Table 1 demonstrate that: a) heating of solutions of H-HA in thestated conditions causes a slight hydrolysis of the long polymer chains(120° C., 10 min MW 9.51·10⁵ Da; 110° C., 10 min MW 1.04·10⁶ Da; 100°C., 10 min MW 1.20·10⁶ Da; 100° C., 40 min MW 9.10·10⁵ Da) withproportional decrease of η; b) heating of solutions of L-HA in thestated conditions causes a slight hydrolysis of the polymer chains (120°C., 10 min MW 2.96·10⁴ Da; 110° C., 10 min MW 3.12·10⁴ Da; 100° C., 10min MW 3.25·10⁴ Da; 100° C., 40 min MW 2.88·10⁴ Da) with proportionaldecrease of η, which after heating is no longer measurable; c) simplemixing in solution of L-HA and H-HA leads, immediately after mixing, toa slight reduction of η, because with L-HA of very low molecular weight(3.3·10⁴ Da) there is commencement of activation, even at roomtemperature, of partial interactions based on hydrogen bonds betweenshort and long chains; d) heating of a solution that contains H-HA andL-HA simultaneously leads to quantitative rupture of the hydrogen bonds,causing loss of the conditions of cooperativeness previously existingbetween the long chains and between the short chains; e) in thesubsequent cooling phase, if both short and long chains are present insolution, cooperative interactions with hydrogen bridges can be randomlyrestored between short and long chains, giving rise to hybrid systemsstabilized by cooperative interactions; f) separate heating of solutionsof H-HA and L-HA and their subsequent mixing after the cooling phasedoes not give rise to formation of cooperative hybrids, but to behavioursimilar to that described at letter c); g) the L/H-HA hybrid, in theabsence of interactions with other molecules or surfaces, remains stableat room temperature because, even if the hydrogen bonds are openedrandomly, the presence of a multiplicity of such interactions along thechains keeps the structural elements responsible for bonding at asuitable distance for its reformation; h) the higher the temperature towhich the H-HA+L-HA mixture is submitted or the longer the exposuretime, the more effective is the formation of the cooperative hybrid.

EXAMPLE 2 Production of Cooperative Hybrid L/H-HA Complexes withDifferent Composition

Cooperative hybrid L/H-HA complexes of different composition areprepared by dissolving H-HA (MW 1.4·10⁶ Da; Mw/Mn 1.5) and L-HA (MW3.3·10⁴ Da; Mw/Mn 1.8) in 100 mL of water, as shown in Table 2. Theresultant solutions are submitted to the following thermal cycle inautoclave: from 25° C. to 120° C. in 10 min, for 10 min at 120° C., from120° C. to 25° C. in 10 min. The dynamic viscosity of the samples, theMW and the polydispersity index Mw/Mn of L-HA, H-HA and L/H-HA aredetermined as described in example 1. The data in Table 2 demonstratethe dependence of the viscosity of L/H-HA cooperative complexes on theL-HA/H-HA ratio: the higher the ratio, the lower the viscosity.

TABLE 2 Sample of L/H-HA L-HA H-HA Treatment (L-HA/H-HA (g in 100 mL of120° C.-10 min w/w) water) η* (Pa · s) η_(H-HA)/η_(L/H-HA) 0.0 0.0 1.05.632 — 0.5 0.5 1.0 0.068 82.8 1.0 1.0 1.0 0.038 148.2 1.5 1.5 1.0 0.033170.7 *The measurements of η are taken immediately after mixing.

Table 2—Measurement of the dynamic viscosity η of cooperative hybridL/H-HA complexes with different L-HA/H-HA ratio. The concentration ofH-HA (MW 1.4·10⁶ Da; Mw/Mn 1.5) is kept constant at 1% w/v, while thatof L-HA (MW 3.3·10⁴ Da; Mw/Mn 1.8) varies from 0 to 1.5% w/v. Thethermal treatment cycle in autoclave envisages a heating phase of 10 minfrom 25° C. to T_(max), remaining at T_(max) for a specified time and acooling phase from T_(max) to 25° C. in 10 min. The measurements of ηare taken immediately after the thermal treatment.

EXAMPLE 3 Production of Cooperative Hybrid L/H-HA Complexes Using L-HAof Different Molecular Weight

Aqueous solutions of H-HA (MW 1.4·10⁶ Da; Mw/Mn 1.5), L-HA (MW 3.3·10⁴Da; Mw/Mn 1.8) and L-HA (MW 2.2·10⁵ Da; Mw/Mn 1.7) are prepared at 2%w/v in distilled water, which are used for preparing the varioussolutions given in Table 3. The resultant solutions are submitted to thefollowing thermal cycle in autoclave: from 25 to 120° C. in 10 min, for10 min at 120° C., from 120 to 25° C. in 10 min. The dynamic viscosity ηof the samples, the MW and the polydispersity index Mw/Mn of L-HA, H-HAand L/H-HA are determined as described in example 1.

The data in Table 3 demonstrate that, all other parameters being equal,the lower the MW of L-HA in the cooperative hybrid, the greater is thedecrease of η. On comparing the values of η of L/H-HA hybrid complexesusing L-HA with MW 3.3·10⁴ Da or 2.20·10⁵ Da the value of the ratioη_(H-HA)/η_(L/H-HA) increases about 50-fold.

TABLE 3 Thermal Sol. 2% p/v treatment (mL) H₂O 120°; 10 min η_(H-HA)/Sample H-HA L-HA (mL) η (Pa · s) η_(L/H-HA) H-HA 100 0 100 5.632 — L-HA3.3 · 10⁴Da 0 100 100 <0.001 — L-HA 2.2 · 10⁵Da 0 100 100 0.016 — L/H-HA3.3 · 10⁴Da 100 100 0 0.038 148.2 L/H-HA 2.2 · 10⁵Da 100 100 0 1.771 3.0

Table 3—Measurement of the dynamic viscosity η of cooperative hybridL/H-HA complexes with L-HA/H-HA ratio of 1 w/w, constructed with L-HA ofdifferent MW. Aqueous solutions of H-HA (MW 1.4·10⁶ Da; Mw/Mn 1.5), L-HA(MW 3.3·10⁴ Da; Mw/Mn 1.8) and L-HA (MW 2.2·10⁵ Da; Mw/Mn 1.7) areprepared at 2% w/v in distilled water, which are used for preparing thevarious solutions given in the table. The resultant solutions aresubmitted to the following thermal cycle in autoclave: from 25 to 120°C. in 10 min, for 10 min at 120° C., from 120 to 25° C. in 10 min.

EXAMPLE 4 Kinetic Analysis of Formation of Cooperative Hybrid L/H-HAComplexes With and Without Thermal Cycle

Aqueous solutions of H-HA (MW 1.4·10⁶ Da; Mw/Mn 1.5), L-HA (MW 3.3·10⁴Da; Mw/Mn 1.8) and L-HA (MW 2.2·10⁵ Da; Mw/Mn 1.7) are prepared at 2%w/v in distilled water, which are used for preparing the varioussolutions given in Table 4. Half of the resultant solutions aremaintained at temperature and the other half are first submitted to thefollowing thermal cycle in autoclave: from 25 to 120° C. in 10 min, for10 min at 120° C., from 120 to 25° C. in 10 min and are then maintainedat room temperature. The dynamic viscosity η is measured over time, forboth series of samples. The MW, the polydispersity index Mw/Mn of L-HA,H-HA and L/H-HA and the dynamic viscosity η of the samples aredetermined as described in example 1.

The data in Table 4 demonstrate that: a) when MW of L-HA is of the orderof 10⁴ Da, formation of L/H-HA cooperative complexes begins, even ifslowly, even at room temperature, because the lower cooperativeness thatexists between the short chains of L-HA allows these to compete in thecooperative interactions existing between the long chains of H-HA,giving rise to the formation of hybrid systems; b) for this reason thesolutions obtained by mixing, at room temperature, H-HA and L-HA with MWof the order of 10⁴ Da, display a dynamic viscosity that varies overtime; c) conversely, the thermal treatment generates, in a few minutes,cooperative hybrid complexes, which once they reach a condition ofequilibrium do not display a change in their dynamic viscosity overtime; d) when the MW of L-HA is of the order of 10⁵, in the absence ofthermal treatment, simple mixing of the two solutions does notsignificantly alter their dynamic viscosity over time, owing to thestrong cooperativeness preexisting between the chains of L-HA, whichprevents their interaction with the chains of H-HA.

TABLE 4 120°; 10′ Without thermal treatment Time (days) 0 10 0 4 14 24Sample η (Pa · s) H-HA + L-HA 0.04 0.04 19.01 6.91 1.05 0.68 3.3 · 10⁴DaH-HA + L-HA 1.77 1.77 24.03 23.71 22.41 21.01 2.20 · 10⁵Da

Table 4—Kinetics of the dynamic viscosity η of cooperative hybrid L/H-HAcomplexes with L-HA/H-HA ratio of 1 w/w, constructed with L-HA ofdifferent MW. Aqueous solutions of H-HA (MW 1.4·10⁶ Da; Mw/Mn 1.5), L-HA(MW 3.3·10⁴ Da; Mw/Mn 1.8) and L-HA (MW 2.2·10⁵ Da; Mw/Mn 1.7) areprepared at 2% w/v in distilled water, which are used for preparing thevarious solutions given in the table. Half of the resultant solutionsare maintained at temperature and half are first submitted to thefollowing thermal cycle in autoclave: from 25 to 120° C. in 10 min, for10 min at 120° C., from 120 to 25° C. in 10 min and are then maintainedat room temperature.

EXAMPLE 5 Preparation of Cooperative Hybrid L/H-HA Complexes in theSolid State by Precipitation from Solution Containing Them

The aqueous solution of the cooperative hybrid L/H-HA complex, obtainedas described in example 1 with a thermal cycle that envisages exposureto a T_(max) of 120° C. for 10 min, is treated with 2 volumes ofanhydrous ethanol, added slowly and with stirring. A white pulverulentprecipitate is obtained, which sediments rapidly and can be dried undervacuum with heating. The process leads to formation of a white drypowder, at a yield of 99% relative to the theoretical value. Thecooperative hybrid L/H-HA complex in powder, if dissolved in water at aconcentration of 1% w/w, gives a solution that has the same value ofdynamic viscosity η as the solution precipitated initially.

EXAMPLE 6 Preparation of Cooperative Hybrid L/H-HA Complexes in theSolid State by Lyophilization of Solutions Containing Them

The aqueous solution of the cooperative hybrid L/H-HA complex, obtainedas described in example 1 with a thermal cycle that envisages exposureto a T_(max) of 120° C. for 10 min is lyophilized. A spongy mass isobtained, which is easily transformed into a white powder by mechanicaltreatment. The yield of lyophilized powder coincides with thetheoretical value. The cooperative hybrid L/H-HA complex lyophilized inpowder, if dissolved in water at a concentration 1% w/w, gives asolution that has the same value of dynamic viscosity η as the solutionprecipitated initially.

EXAMPLE 7 Preparation of Cooperative Hybrid C/H-HA and CS/H-HA Complexes

Aqueous solutions of H-HA (MW 1.4·10⁶ Da; Mw/Mn 1.5), chondroitin (C; MW6.6·10⁴ Da; Mw/Mn 1.4) and chondroitin sulphate (CS; MW 3.8·10⁴ Da;Mw/Mn 1.4) are prepared at 2% w/v in distilled water, which are used forpreparing the various solutions given in Table 5. The resultantsolutions are submitted to the following thermal cycle in autoclave:from 25 to 120° C. in 10 min, for 10 min at 120° C., from 120 to 25° C.in 10 min. The dynamic viscosity η of the samples, the MW and thepolydispersity index Mw/Mn of L-HA, H-HA and L/H-HA are determined asdescribed in example 1.

The data in Table 5 demonstrate that: a) both chondroitin andchondroitin sulphate, with MW of the order of magnitude of 10⁴ Da, giverise by thermal treatment to the formation of stable cooperative hybridC/H-HA and CS/H-HA complexes, characterized by a low value of dynamicviscosity η; b) simple mixing of solutions of C and CS with that of H-HAdoes not produce significant changes in dynamic viscosity η; c) theη_(pre)/η_(post) ratio of the C/H-HA complex is about double that of theCS/H-HA complex.

TABLE 5 Thermal post- treatment Sol. 2% p/v Thermal 120°; (mL) H₂Opretreatment 10 min Sample H-HA L-HA (mL) η (Pa · s) η_(pre)/η_(post)H-HA 100 0 100 21.321 5.632 — C 0 100 100 0.004 0.002 — CS 0 100 1000.002 0.001 — C/H-HA 100 100 0 22.832 0.873 27.3 CS/H-HA 100 100 022.915 1.728 13.2

Table 5—Measurement of the dynamic viscosity η of cooperative hybridC/H-HA and CS/H-HA complexes. Aqueous solutions of H-HA (MW 1.4·10⁶ Da;Mw/Mn 1.5), C (MW 6.6·10⁴ Da; Mw/Mn 1.4) and CS (MW 3.8·10⁴ Da; Mw/Mn1.4) are prepared at 2% w/v in distilled water, which are used forpreparing the various solutions given in the table. The resultantsolutions are submitted to the following thermal cycle in autoclave:from 25 to 120° C. in 10 min, for 10 min at 120° C., from 120 to 25° C.in 10 min.

EXAMPLE 8 Use of Cooperative Hybrid L/H-HA Complexes in the Field ofBiorevitalization

4g of the cooperative hybrid complex obtained as described in example 1is dissolved in 100 mL of saline, heating at 120° C. for 10 min, andthen drying the complex by lyophilization, as described in example 5.The hyaluronic acid used is of pharmaceutical grade for injection andall the manipulations are carried out in conditions that guaranteesterility and apyrogenicity of the solution. The solution containing 40mg/mL of L/H-HA complexes is introduced into 1 mL syringes fitted with agauge 30 needle. The treatment of biorevitalization of the face isconducted on 10 informed volunteers, who have obvious signs of cutaneousageing of the face. The experimental design envisages that each subjectundergoes an identical treatment of biorevitalization by subcutaneousmicroinjection on the right side of the face with the formulation of theinvention (1 mL) and on the left side with a primary product alreadymarketed (1 mL). The results obtained, objectivized instrumentally,demonstrate the superiority of the treatment with the stable cooperativeL/H-HA complex, both in terms of quality and duration of the treatment.

EXAMPLE 9 Use of Cooperative Hybrid L/H-HA Complexes in the Field ofViscosupplementation

4g of the cooperative hybrid complex obtained as described in example 1is dissolved in 100 mL of saline, heating at 120° C. for 10 min, andthen drying the complex by lyophilization, as described in example 5.The hyaluronic acid used is of pharmaceutical grade for injection andall the manipulations are carried out in conditions that guaranteesterility and apyrogenicity of the solution. The solution containing 40mg/mL of L/H-HA complexes is introduced into 1 mL syringes fitted with agauge 30 needle. The viscosupplementation treatment is conducted on 5informed volunteers, with a bilateral knee disorder, the therapeuticindication being infiltration of hyaluronic acid in the joint. Theexperimental design envisages that each subject receives identicaltreatment of viscosupplementation in the right joint with theformulation of the invention (1 mL) and in the left joint with a primaryproduct already marketed (1 mL). The results obtained, objectivizedinstrumentally, demonstrate the superiority of the treatment with thestable cooperative L/H-HA complex, both in terms of rapid reduction ofpain and efficacy of resolution of the pathological condition.

1. Stable, hybrid cooperative L/H-HA complexes prepared by submitting tothermal treatment, at temperature comprised between 80° and 160°,solutions containing simultaneously L-HA hyaluronic acid or hyaluronansand H-HA hyaluronic acid or hyaluronans wherein the molecular weight ofL-HA is comprised between 1×10⁴ to 1×10⁶ Da and that of H-HA is given bythe formula MW_(H-HA)≧_(MWL-HA)/0.9.
 2. Stable complexes according toclaim 1 wherein said temperature is comprised between 100° and 120° C.3. Hybrid cooperative complexes according to claim 1, wherein H-HA andL-HA are present in relative quantities comprised between 0.1 and
 10. 4.L/H-HA complexes according to claim 1 having a viscosity from 1.1 to200-fold less than that of a solution containing the H-HA hyaluronicacid alone used for forming the complex.
 5. L/H-HA complexes accordingto claim 4, wherein other chemical species in addition to solvent andhyaluronic acid are also present in the solution.
 6. L/H-HA complexesaccording to claim 1, wherein the solvent used is water.
 7. L/H-HAcomplexes according to claim 1, wherein the thermal treatment is carriedout in autoclave.
 8. L/H-HA complexes according to claim 1, wherein thethermal treatment includes keeping the sample at the maximal temperaturefor times which are from 10 seconds to 2 hrs.
 9. L/H-HA complexesaccording to claim 8, wherein the thermal treatment includes reachingthe maximal temperature over times from 1 min to 1 h.
 10. L/H-HAcomplexes according to claim 8 wherein the thermal treatment includescooling the solution, after exposure at the maximal temperature, overtimes from 1 min to 1 h.
 11. L/H-HA complexes according to claim 1,prepared in the solid state by precipitation of the solutions containingthem.
 12. L/H-HA complexes according to claim 11, wherein theprecipitation is obtained by adding a compound miscible with thesolution and acting as a non-solvent for the complex.
 13. L/H-HAcomplexes according to claim 12, wherein the non-solvent compound forthe complex is a water-miscible organic solvent, selected from the groupconsisting of acetone, methanol, ethanol, propanol, isopropanol andbutanol.
 14. L/H-HA complexes according to claim 1, prepared by removingthe solvent by means of vacuum evaporation, spray-drying orfreeze-drying in the dry state.
 15. Hybrid cooperative complexesaccording to claim 1, wherein the low molecular weight hyaluronic acidis replaced by other low molecular weight polysaccharides, such aschondroitin, chondroitin sulphate, dextrins, cyclodextrins, dextrans.16. Hybrid cooperative complexes according to claim 15 wherein by lowmolecular weight of polysaccharides is meant less than one million ofDa.
 17. Method of treating patients in need thereof with hybridcooperative complexes of claim 1 comprising: administering to saidpatients said hybrid cooperative complexes as a replacement for highmolecular weight hyaluronic acid in all clinical treatments based onsaid high weight hyaluronic acid and treating said patients in need. 18.Method for intradermal bio-revitalizing treatment with hybridcooperative complexes according to claim 1 comprising: administeringsaid hybrid cooperative complexes to patients in need thereof andtreating said patients.
 19. Method for intra-articularviscosupplementation with hybrid cooperative complexes according toclaim 1 comprising administering said hybrid cooperative complexes topatients in need thereof and treating said patients.
 20. Method forintra-bladder treatments of cystitis with hybrid cooperative complexesaccording to claim 1 comprising: administering said hybrid cooperativecomplexes to patients in need thereof and treating said patients. 21.Method of treating vaginal inflammatory diseases with hybrid cooperativecomplexes according to claim 1 comprising administering said hybridcooperative complexes to patients in need thereof and treating saidpatients.
 22. Method of treating alveolar diseases with hybridcooperative complexes according to claim 1 comprising administering saidhybrid cooperative complexes to patients in need thereof and treatingsaid patients.
 23. Method of treating oral disease with hybridcooperative complexes according to claim 1 comprising administering saidhybrid cooperative complexes to patients in need thereof and treatingsaid patients.